Project Summary Glioblastomas are the most common form of primary malignant brain tumors and affect over 15,000 new individuals in the United States annually. With surgical resection, chemotherapy and radiotherapy the prognosis for patients is only at 15 months; and while checkpoint inhibitor therapy has provided unprecedented clinical benefit for other cancers, its effect on brain tumors is unclear. This is attributed to the unique immune environment in the central nervous system, with the draining lymphatic vasculature only being (re)identified few years ago. Before this discovery, the brain was often thought to be immune-privileged, not having traditional efferent and afferent immune cell trafficking and unable to evoke similar immune responses as peripheral organs. But even with the characterization of this CNS lymphatic network, little is known about what makes the meningeal lymphatics unique from other lymphatic networks in the body, especially during chronic inflammatory states such as the presence of a tumor. We hypothesize that at homeostasis, the CNS immunosurveillance may not be sufficient to evoke an immune response; but by increasing the lymphatic drainage to allow for more T cell priming, we can generate an adequate inflammatory response against tumors. In Aim 1, we will identify which cells are required for immunosurveillance and rejection of brain tumors. By introducing VEGFC-AAV into the cisterna magna of mice, the meningeal lymphatic network proliferates, prompting a strong immune response against orthotopic brain tumor models. With depletion antibodies and various ko mice, we will identify which cell types are required for tumor rejection in these mice through survival studies, flow cytometry and immunofluorescent staining. In Aim 2, we will investigate whether upregulation of VEGF-C can be used either as a monotherapy, or to potentiate current immunotherapy strategies. To accomplish this, we will use mRNA gene therapy to transiently increase VEGF-C in mice after brain tumor implantation. mRNA has several advantages to other gene therapy strategies-including its cost, efficiency and controlled protein expression kinetics in mammalian systems. We designed the mRNA enhancement strategy to mitigate any potential long-term harm of VEGF-C expression, while allowing VEGF-C to synergistically work with checkpoint inhibitor therapies such as, PDL-1, CTLA-4 and 4-1BB in treating GBM. Finally, in Aim 3 dural lymphatic vessels from postmortem GBM patients or from unrelated diseases will be evaluated. The dural lymphatics have only recently been identified in mice, and there are still many questions regarding its role in patients. We will evaluate what effects the chronic inflammatory state of a brain tumor has on the meningeal lymphatics by staining GBM patient?s dura (postmortem) and compared to those who died of unrelated causes. These three aims will help support our hypotheses of how immunosurveillance occurs in the CNS and help us design better treatment for GBM. We expect that our findings will uncover new strategies to make the CNS vulnerable to immunotherapy and help gain new insight to understanding cancer immunology. !!